Heart failure (HF) is a devastating disease with poor prognosis. Pathologic cardiac remodeling is characterized by progressive hypertrophy, apoptosis and fibrosis. Fibrosis exacerbates cardiac remodeling, arrhythmias, sudden cardiac death, and decreased ventricular compliance; emerging evidence suggests that cardiac fibroblasts (CFs) play a key role in these processes. To date, there are no therapies that target fibrosis. Elucidating the role(s) of CF in pathologic remodeling may hold substantial therapeutic potential. CF studies have been limited by the lack of genetic reagents in vivo. We are collaboratively characterizing two novel mouse models (PeriostinMerCreMer and Tcf21MerCreMer knock-in mice) that permit targeted and inducible manipulation of CF function/viability to directly determine the mechanistic basis of cardiac fibrosis. Chronic G- protein coupled receptor (GPCR) stimulation in HF elicits pathologic upregulation of GPCR kinase 2 (GRK2) that is recruited to membrane G?? subunits to desensitize agonist-occupied GPCRs. We and others have demonstrated therapeutic potential for inhibiting G??-GRK2 interaction in HF. Our novel, small molecule G??- GRK2 inhibitor, gallein, attenuates pathologic cardiac remodeling, normalizes GPCR signaling and reduces interstitial myocardial fibrosis in HF. To explore a fibroblast-restricted role for GRK2 in myocardial fibrosis, GRK2fl/fl mice have been bred with POSTNMerCreMer and Tcf-21MerCreMer. Our exciting preliminary data suggest that the GRK2fl/fl-POSTNMerCreMer mice are significantly protected following cardiac ischemia/reperfusion (I/R) injury, with no further protection conferred by gallein. Interestingly, GRK2fl/fl-POSTNMerCreMer mice appeared to be more protective than GRK2fl/fl-?MHCMerCreMer, suggesting that greater cardioprotection may be provided via the CF than previously thought. Worsening renal function and fibrosis often accompanies HF progression; it is a strong predictor of mortality in advanced HF patients (cardio-renal syndrome 2, CRS2) but remains poorly understood. TAC or I/R injury resulted in substantial kidney fibrosis and dysfunction in our studies; systemic gallein attenuated CRS2 as well as renal dysfunction and fibrosis following acute renal I/R injury. Our hypothesis is that G??-GRK2 plays an important role in pathologic CF activation in HF progression and kidney dysfunction and that its inhibition holds therapeutic promise for HF and CRS2. To address our hypothesis, we propose the following Aims: Aim 1: Determine the therapeutic efficacy and specificity of G??-GRK2 inhibition or GRK2 ablation in a clinically-relevant HF model in wild type and inducible CF or CM GRK2-/- mice (+/- Gallein). Aim 2: Elucidate the cellular mechanisms underlying G??-GRK2 inhibition in fibroblasts and validate G??-GRK2 inhibition as a therapeutic target in both mouse and human cardiac fibrosis and HF. Aim 3: Determine the role of G??-GRK2 signaling in renal fibrosis and CRS2. We believe this proposal, with G??-GRK2 inhibitory compounds, validated in CF-restricted GRK2-/- mice, holds therapeutic promise for HF. The principle may also extend to CRS2 and other fibrotic diseases.